Phanerochaete chrysosporium β-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate β-glucosidases and an extracellular β-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K_m for p-nitrophenyl-β-glucoside is 1.6 × 10⁻⁴ M; the K_i for glucose, a competitive inhibitor, is 5.0 × 10⁻⁴ M. The K_m for cellobiose is 5.3 × 10⁻⁴ M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K_m for p-nitrophenyl-β-glucoside is 1.1 × 10⁻⁴ M. The molecular weight of this enzyme is ~410,000. Both enzymes have an optimal temperature of 45°C and an E_act of 9.15 kcal (ca. 3.83 × 10⁴ J). The pH optima, however, were ~7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre-Steady-State Study

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k_cat and k_cat/K_m reflect the ionization of two groups with pK_a values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K_m are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10⁻¹⁰ molar at the optimum pH value (approximately 7.5).     

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (k_cat/K_m) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (k_cat/K_m >10⁵ s⁻¹ m⁻¹ for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (K_iS ∼100 μm). By comparison, the cholesterol-degrading KshA_Mtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshA_Mtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate''s C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.     

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19_Mtb) transformed 3-oxo-4-cholesten-26-oate (k_cat/K_m = 0.33 × 10⁵ ± 0.03 × 10⁵ M⁻¹ s⁻¹) and represents orthologs that activate the C₈ side chain of cholesterol. Both CasG_RHA1 and FadD17_Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C₅ side chain of cholate, while FadD17 and its orthologs appear to activate the C₅ side chain of one or more cholesterol metabolites. CasI_RHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C₃ side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (k_cat/K_m = 2.4 × 10⁵ ± 0.1 × 10⁵ M⁻¹ s⁻¹ and 3.2 × 10⁵ ± 0.3 × 10⁵ M⁻¹ s⁻¹, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.     

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6¹,6³- and 6¹,6⁴-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the k_cat and K_m values for transglycosylation were 1.78 × 10³ s⁻¹ and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10³ s⁻¹ and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the k_cat/K_m ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

An Inverse Power-Law Distribution of Molecular Bond Lifetimes Predicts Fractional Derivative Viscoelasticity in Biological Tissue

Viscoelastic characteristics of many materials falling under the category of soft glassy substances, including biological tissue, often exhibit a mechanical complex modulus Y(ω) well described by a fractional derivative model: Y(ω) = E(iω/ϕ)^k, where E = a generalized viscoelastic stiffness; i = (−1)^1/2; ω = angular frequency; ϕ = scaling factor; and k = an exponent valued between 0 and 1. The term “fractional derivative” refers to the value of k: when k = 0 the viscoelastic response is purely elastic, and when k = 1 the response is purely viscous. We provide an analytical derivation of the fractional derivative complex modulus based on the hypothesis that the viscoelastic response arises from many intermittent molecular crosslinks, whose lifetimes longer than a critical threshold lifetime, t_crit, are distributed with an inverse power law proportional to t^-(k+2). We demonstrate that E is proportional to the number and stiffness of crosslinks formed at any moment; the scaling factor ϕ is equivalent to reciprocal of t_crit; and the relative mean lifetime of the attached crosslinks is inversely proportional to the parameter k. To test whether electrostatic molecular bonds could be responsible for the fractional derivative viscoelasticity, we used chemically skinned human skeletal muscle as a one-dimensional model of a soft glassy substance. A reduction in ionic strength from 175 to 110 mEq resulted in a larger E with no change in k, consistent with a higher probability of interfilament molecular interactions. Thick to thin filament spacing was reduced by applying 4% w/v of the osmolyte Dextran T500, which also resulted in a larger E, indicating a greater probability of crosslink formation in proportion to proximity. A 10°C increase in temperature resulted in an increase in k, which corresponded to a decrease in cross-bridge attachment lifetime expected with higher temperatures. These theoretical and experimental results suggest that the fractional derivative viscoelasticity observed in some biological tissue arises as a mechanical consequence of electrostatic interactions, whose longest lifetimes are distributed with an inverse power law.     

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYME·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C–H BOND CLEAVAGE*

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both k_cat and k_cat/K_m, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (k_on 2.4 × 10⁷ m⁻¹ s⁻¹) is only ∼2-fold greater than the catalytic efficiency (k_cat/K_m = 1.3 × 10⁷ m⁻¹ s⁻¹) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.     

Cloning,purification, kinetic and anion inhibition studies of a recombinant β-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

A β-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAβ has a significant catalytic activity for the physiological reaction, CO₂ + H₂O ⇋ HCO₃⁻ + H⁺ with a k_cat of 1.1 × 10⁵ s⁻¹ and a k_cat/K_m of 7.58 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by acetazolamide (K_I of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAβ at millimolar concentrations, but sulfamide (K_I of 81 µM), N,N-diethyldithiocarbamate (K_I of 67 µM) and sulphamic acid (K_I of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAβ is subsequently proposed as a new drug target for which effective inhibitors can be designed.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Ficin-Catalyzed Reactions. Hydrolysis of alpha-N-Benzoyl-l-Arginine Ethyl Ester and alpha-N-Benzoyl-l-Argininamide

The effect of pH on the hydrolysis of α-N-benzoyl-l-arginine ethyl ester (BAEE) and α-N-benzoyl-l-argininamide (BAA) by a proteolytic enzyme component purified from Ficus carica var. Kadota latex has been studied in detail over the pH range of 3 to 9.5. k_cat (lim) values for the hydrolysis of BAEE and BAA were essentially identical (5.20 and 5.01 sec⁻¹, respectively at 30°). k_cat values for hydrolysis of BAEE and BAA were dependent on prototropic groups with apparent pK values of 4.24 and 8.53 and 4.10 and 8.59, respectively. k_cat (lim) values for tht hydrolysis of BAEE and BAA were essentially identical (5.20 and groups of pK 4.33 and 8.60 and 4.55 and 8.51, respectively. Thus the pH optimum is 6.5 for both substrates. K_m (app) values for BAEE and BAA were 3.32 × 10⁻²m and 6.03 × 10⁻²m respectively over the pH range of 3.9 to 8.0. These data are interpreted in terms of the involvement of a carboxyl and a sulfhydryl group in the active center of the enzyme. The data do not support the concept that deacylation of the acyl-enzyme is completely the rate controlling step in the hydrolyses. Rather, it appears that the magnitude of k₂ and k₃ are not greatly different.     

Gender-specific genetic associations of polymorphisms in ACE,AKR1C2, FTO and MMP2 with weight gain over a 10-year period

Weight gain, when it leads to overweight or obesity, is nowadays one of the major health problems. ACE, FTO, AKR1C2, TIMP4 and MMP2 genes have been implicated in previous studies on weight regulation. This study investigated the contribution of polymorphisms in these five candidate genes to the risk of weight gain over a 10-year time period. Two groups were selected from participants of the Doetinchem cohort study who were followed over a 10-year period: A stable weight group (±2 kg/10 year; n = 259) and a weight gainers group who increased their body weight by roughly 10 % (>8 kg/10 year; n = 237). Starting BMI was between 20 and 35 kg/m² and baseline age between 20 and 45 years. Selected SNPs and insert/deletion in candidate genes were measured in each group. In men, the allelic distribution of FTO rs9939609 (χ²p = 0.005), ACE rs4340 (χ²p = 0.006) and AKR1C2 rs12249281 (χ²p = 0.019) differed between the weight stable and weight gainers group. Interaction between FTO rs9939609 and ACE rs4340 was observed. In women, the allelic distribution of MMP2 rs1132896 differed between the weight stable and weight gainers group (χ²p = 0.00001). The A-allele of FTO was associated with a 1.99× higher risk of gaining weight in men (OR 1.99, p = 0.020), while in women, the C-allele of MMP2 was associated with a 2.50× higher risk of weight gain (OR 2.50, p = 0.001) over the 10-year period. We found that FTO in men and MMP2 in women are associated with weight gain over a 10-year follow-up period.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0434-2) contains supplementary material, which is available to authorized users.     

Role of Sulfate Reduction Versus Methanogenesis in Terminal Carbon Flow in Polluted Intertidal Sediment of Waimea Inlet, Nelson, New Zealand

An investigation of the terminal anaerobic processes occurring in polluted intertidal sediments indicated that terminal carbon flow was mainly mediated by sulfate-reducing organisms in sediments with high sulfate concentrations (>10 mM in the interstitial water) exposed to low loadings of nutrient (equivalent to <10² kg of N · day⁻¹) and biochemical oxygen demand (<0.7 × 10³ kg · day⁻¹) in effluents from different pollution sources. However, in sediments exposed to high loadings of nutrient (>10² kg of N · day⁻¹) and biochemical oxygen demand (>0.7 × 10³ kg · day⁻¹), methanogenesis was the major process in the mediation of terminal carbon flow, and sulfate concentrations were low (≤2 mM). The respiratory index [¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism, a measure of terminal carbon flow, was ≥0.96 for sediment with high sulfate, but in sediments with sulfate as little as 10 μM in the interstitial water, respiratory index values of ≤0.22 were obtained. In the latter sediment, methane production rates as high as 3 μmol · g⁻¹ (dry weight) · h⁻¹ were obtained, and there was a potential for active sulfate reduction.     

Intracellular Biopotentials During Static Extracellular Stimulation

Two properties of the intracellular potentials and electric fields resulting from static extracellular stimulation are obtained for arbitrarily shaped cells. First, the values of intracellular potential are shown to be bounded by the maximum and minimum values of extracellular potential on the surface of the cell. Second, the volume average of the magnitude of intracellular electric field is shown to have an upper bound given by the ratio of the magnitude of the largest extracellular potential difference on the surface of the cell to a generalized length constant λ = [σ_intraV_cell/(σ_memb A_cell)]^1/2, where V_cell and A_cell are the volume and surface area of the cell, σ_intra is the intracellular conductivity (reciprocal ohms per centimeter), and σ_memb is the membrane conductivity (reciprocal ohms per square centimeter). The use of the upper bound on the volume average of the magnitude of intracellular electric field as an estimate for intracellular isopotentiality is discussed and the use of the generalized length constant for electrically describing arbitrary cells is illustrated for cylindrical- and spheroidal-shaped cells.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

The Anisotropic Elastic Properties of the Sarcolemma of the Frog Semitendinosus Muscle Fiber

Tension and curvature of the sarcolemmal tube of the frog muscle fiber were measured at different extensions and were used to calculate the anisotropic elastic properties of the sarcolemma. A model was derived to obtain the four parameters of the elasticity matrix of the sarcolemma. Sarcolemmal thickness was taken as 0.1 μm. Over the range of reversible sarcolemmal tube extension, the longitudinal elastic modulus E_L = 6.3 × 10⁷ dyn/cm², the circumferential modulus E_c = 0.88 × 10⁷ dyn/cm², the longitudinal Poisson's ratio σ_L = 1.2, and the circumferential Poisson's ratio σ_c = 0.18. At tubular rest length E_L = 1.2 × 10⁷ dyn/cm². The sarcolemma is less extensible in the longitudinal direction along the fiber axis than in the circumferential direction. It can be extended reversibly to 48% of its rest length, equivalent to extending the intact fiber from a sarcomere length of 3 μm to about 4.5 μm. The sarcolemma does not contribute to intact fiber tension at fiber sarcomere lengths <3 μm, and between 3 and 4 μm its contribution is about 20%. It also exerts a pressure on the myoplasm, which can be calculated by means of the model. The longitudinal elastic modulus of the whole fiber is 1 × 10⁵ dyn/cm² at a sarcomere length of 2.33 μm.     

NO? Binds Human Cystathionine β-Synthase Quickly and Tightly

The hexa-coordinate heme in the H₂S-generating human enzyme cystathionine β-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO^• or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO^• was reported to bind at the CBS heme with very low affinity (K_d = 30–281 μm). This discrepancy was herein reconciled by investigating the NO^• reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO^• binds tightly to the ferrous CBS heme, with an apparent K_d ≤0.23 μm. In line with this result, at 25 °C, NO^• binds quickly to CBS (k_on ∼ 8 × 10³ m⁻¹ s⁻¹) and dissociates slowly from the enzyme (k_off ∼ 0.003 s⁻¹). The observed rate constants for NO^• binding were found to be linearly dependent on [NO^•] up to ∼ 800 μm NO^•, and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO^• quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO^•. The novel findings reported here shed new light on CBS regulation by NO^• and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO^•, CO, and H₂S in cell signaling.